PAI-1over expression in diabetes may be central to all of the major complications of diabetes; retinopathy, nephropathy, neuropathy, wound healing and macrovascular disease. It is a required component in development, tissue migration, tissue remodeling and cell adhesion as well as being a potent inhibitor of protease activation. Its production is highly disregulated in diabetes due to increased transcription in response to insulin and oxidative stress. It is essential to understand how insulin and oxidative stress activate the PAI-1 promoter and how its activity can be normalized in diabetes. Hyperinsulinemia is one of the key changes in type II diabetes while patients with type I diabetes and treated with insulin are exposed to pharmacological insulin levels. It has recently become appreciated that although cells become resistant to the metabolic effects of insulin, the mitogenic effects of insulin remain insulin responsive. This results in a double defect with glucose transport and metabolism becoming refractory to insulin while transcription of insulin responsive genes is hyperactivated. PAI-1 is one such insulin-activated gene. The PAI-1 promoter is also responsive to oxidative stress that is part of the pathology of diabetes and insulin and oxidative stress combine to produce an additive increase in PAI-1 production. We have defined the insulin response element of the PAI-1 promoter and have determined that the insulin activated transcription factor that activates this element is a Forkhead-related transcription factor. We have also determined that oxidative stress activates an AP-1 response element that is adjacent to the insulin response element. This is accomplished through activation of JNK/SAPK and phosphorylation of c-jun that increases binding of the AP1 complex to the PAI-1 promoter. Insulin and oxidative stress induce an additive activation of transcription. It is important to identify the Forkhead related transcription factor that mediates the insulin response of the promoter and to determine how it is activated by insulin. This proposal sets forth a strategy for accomplishing these goals. [unreadable] [unreadable]